Basic fibroblast growth factor (bFGF) is unstable and rapidly degrades upon storage and during delivery, which hinders its therapeutic applications. Heparin has been shown to protect bFGF from denaturation by heat and acid, and also to prolong its half-life. Thus, the polysaccharide has been used in controlled release systems of bFGF. However, heparin is difficult to modify and exhibits batch-to-batch variability. It also has significant bioactivity in other, non-target biological pathways. It is known that sulfated and sulfonated small molecules and polymers are capable of mimicking heparin and these have been explored for stabilization of the protein. An important consideration when utilizing such polymers is that high doses of heparin (and heparin-mimicking compounds) have biological effects such as bleeding complications due to anticoagulant activity and anti-angiogenic activity. This activity would be particularly deleterious for therapeutic applications. Covalently binding a heparin-mimicking polymer to bFGF would ensure that a therapeutically useful concentration of the polymer is employed. Despite this advantage, conjugates of heparin mimicking polymers and bFGF have not yet been prepared.
bFGF has been conjugated to other proteins and peptides, but not for the purpose of increasing stability. A histidine-tagged bFGF-8b (related to bFGF) has been non-covalently conjugated to a nitriloacetic acid (NTA) Ni2+ modified poly(acrylamide), but this complex is non-covalent, requires the use of a toxic metal, and was found to be less active than bFGF. PEG-polyanions (pentosan polysulfate and dextran sulfate) have also been utilized to increase stability of a related protein keratinocyte growth factor-2 (KGF-2), but these complexes are also non-covalent. Non-covalent conjugates are likely not to be useful in vivo because of the likelihood of detachment of the polymer upon dilution before reaching target sites. Covalent conjugates of poly(ethylene glycol) (PEG) and bFGF have been synthesized, but these either exhibit significant reductions in protein activity and require addition of heparin to stabilize the conjugate or large concentrations of the conjugate need to be utilized. This invention describes the first conjugate between bFGF and a heparin-mimicking polymer. The conjugate not only retains bioactivity, but is stable to environmental and therapeutically relevant stressors including storage, heat, enzymes, and reduced pH.